Anti-ErbB3 Antibodies and Uses Thereof

ABSTRACT

The present invention provides antibodies that bind to ErbB3 and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human ErbB3. In certain embodiments, the antibodies of the present invention block the interaction of ErbB3 with an ErbB3 ligand such as neuregulin 1. The antibodies of the invention are useful for the treatment of various cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/623,885,filed Sep. 21, 2012, which claims the benefit under 35 U.S.C. §119(e) ofU.S. provisional application Nos. 61/541,312, filed on Sep. 30, 2011;61/557,460, filed on Nov. 9, 2011; and 61/614,565, filed on Mar. 23,2012, the disclosures of which are herein incorporated by reference intheir entireties.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“447339-Sequence.txt”, created on Jun. 18, 2014 and containing 183,959bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for human ErbB3.

BACKGROUND

ErbB3 (also known as HER3) is a member of the ErbB/HER family ofreceptor tyrosine kinases (RTKs). Other members of this family includeEGFR (also known as ErbB1 or HER1), ErbB2 (also known as HER2 or Neu),and HER4. ErbB receptors regulate cell proliferation, survival anddifferentiation by activating intracellular signaling cascades that leadto alterations in gene expression.

ErbB receptors are activated by the formation of either homo- orheterodimers. For example, when ErbB3 is co-expressed with ErbB2, anactive heterodimeric signaling complex is formed. ErbB3 dimer formationis promoted by its ligand binding. Neuregulin 1 (NRG1) is the primaryligand for ErbB3 that promotes homo- or heterodimerization of thereceptor.

ErbB3 has been found to be overexpressed in various cancer types,including breast, gastrointestinal, and pancreatic cancers. Anti-ErbB3antibodies have been shown to inhibit the growth of several human tumorcell lines in mouse xenografts models. Anti-ErbB3 antibodies arementioned in, e.g., U.S. Pat. No. 5,480,968; U.S. Pat. No. 5,968,511; US2004/0197332; U.S. Pat. No. 7,332,580; U.S. Pat. No. 7,705,130; and U.S.Pat. No. 7,846,440. Nonetheless, there is a need in the art for novelErbB3 antagonists, such as anti-ErbB3 antibodies, for the treatment ofcancer and other related disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind human ErbB3. Theantibodies of the invention are useful, inter alia, for inhibitingErbB3-mediated signaling and for treating diseases and disorders causedby or related to ErbB3 activity and/or signaling.

The antibodies of the present invention, according to certainembodiments, block the interaction between ErbB3 and an ErbB3 ligand(e.g., NRG1 and/or NRG2). The antibodies may also possess one or moreadditional biological properties such as, e.g., inducing cell surfaceErbB3 internalization, inhibiting NRG1-stimulated tumor growth in vitro,and/or inhibiting tumor growth in vivo.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

The present invention provides an antibody or antigen-binding fragmentof an antibody comprising a heavy chain variable region (HCVR) having anamino acid sequence selected from the group consisting of SEQ ID NO: 2,18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242,258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 466 and482, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442,458, 474 and 490, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, 322/330, 338/346, 354/362, 370/378, 386/394, 402/410, 418/426,434/442, 450/458, 466/474 and 482/490.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440,456, 472 and 488 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;and a light chain CDR3 (LCDR3) domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96,112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320,336, 352, 368, 384, 400, 416, 432, 448, 464, 480 and 496, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208,216/224, 232/240, 248/256, 264/272, 280/288, 296/304, 312/320, 328/336,344/352, 360/368, 376/384, 392/400, 408/416, 424/432, 440/448, 456/464,472/480 and 488/496.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260,276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468 and 484,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; a heavy chainCDR2 (HCDR2) domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134,150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358,374, 390, 406, 422, 438, 454, 470 and 486, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; a light chain CDR1 (LCDR1) domain having anamino acid sequence selected from the group consisting of SEQ ID NO: 12,28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252,268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476 and492, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a lightchain CDR2 (LCDR2) domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350,366, 382, 398, 414, 430, 446, 462, 478 and 494, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H4H2084P);20-22-24-28-30-32 (e.g. H4H2092P); 36-38-40-44-46-48 (e.g. H4H2094P);52-54-56-60-62-64 (e.g. H4H2098P); 68-70-72-76-78-80 (e.g. H4H2102P);84-86-88-92-94-96 (e.g. H4H2108P); 100-102-104-108-110-112 (e.g.H4H2111P); 116-118-120-124-126-128 (e.g. H4H2114P);132-134-136-140-142-144 (e.g. H4H2132P); 148-150-152-156-158-160 (e.g.,H4H2138P); 164-166-168-172-174-176 (e.g. H4H2140P);180-182-184-188-190-192 (e.g., H4H2143P); 196-198-200-204-206-208 (e.g.H4H2146P); 212-214-216-220-222-224 (e.g. H4H2147P);228-230-232-236-238-240 (e.g. H4H2148P); 244-246-248-252-254-256 (e.g.H4H2151P); 260-262-264-268-270-272 (e.g. H4H2153P);276-278-280-284-286-288 (e.g. H4H2154P); 292-294-296-300-302-304 (e.g.H4H2290P); 308-310-312-316-318-320 (e.g. H1M1819N);324-326-328-332-334-336 (e.g. H2M1821N); 340-342-344-348-350-352 (e.g.H2M1824N); 356-358-360-364-366-368 (e.g. H2M1827N);372-374-376-380-382-384 (e.g. H1M1828N); 388-390-392-396-398-400 (e.g.H2M1829N); 404-406-408-412-414-416 (e.g. H2M1930N);420-422-424-428-430-432 (e.g. H2M1943N); 436-438-440-444-446-448 (e.g.H2M1936N); 452-454-456-460-462-464 (e.g. H2M1937N);468-470-472-476-478-480 (e.g. H2M1938N); and 484-486-488-492-494-496(e.g. H1M1940N).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically binds ErbB3,wherein the antibody or fragment comprises the heavy and light chain CDRdomains contained within heavy and light chain sequences selected fromthe group consisting of SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74,82/90, 98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202,210/218, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330,338/346, 354/362, 370/378, 386/394, 402/410, 418/426, 434/442, 450/458,466/474 and 482/490. Methods and techniques for identifying CDRs withinHCVR and LCVR amino acid sequences are well known in the art and can beused to identify CDRs within the specified HCVR and/or LCVR amino acidsequences disclosed herein. Exemplary conventions that can be used toidentify the boundaries of CDRs include, e.g., the Kabat definition, theChothia definition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In another aspect, the invention provides nucleic acid moleculesencoding anti-ErbB3 antibodies or fragments thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337,353, 369, 385, 401, 417, 433, 449, 465 and 481, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof.

The present invention also provides an antibody or fragment thereofcomprising a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361,377, 393, 409, 425, 441, 457, 473 and 489, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247,263, 279, 295, 311, 327, 343, 359, 375, 391, 407, 423, 439, 455, 471 and478, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207,223, 239, 255, 271, 287, 303, 319, 335, 351, 367, 383, 399, 415, 431,447, 463, 479 and 495, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofwhich further comprises a HCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83,99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307,323, 339, 355, 371, 387, 403, 419, 435, 451, 467 and 483 or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof; a HCDR2 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 5,21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245,261, 277, 293, 309, 325, 341, 357, 373, 389, 405, 421, 437, 453, 469 and485, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; a LCDR1 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203,219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427,443, 459, 475 and 491, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof;and a LCDR2 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141,157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365,381, 397, 413, 429, 445, 461, 477 and 493, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof.

According to certain embodiments, the antibody or fragment thereofcomprises the heavy and light chain CDR sequences encoded by the nucleicacid sequences of SEQ ID NOs: SEQ ID NOs: 1 and 9 (e.g. H4H2084P), 17and 25 (e.g. H4H2092P), 33 and 41 (e.g. H4H2094P), 49 and 57 (e.g.H4H2098P), 65 and 73 (e.g. H4H2102P), 81 and 89 (e.g. H4H2108P), 97 and105 (e.g. H4H2111P), 113 and 121 (e.g. H4H2114P), 129 and 137 (e.g.H4H2132P), 145 and 153 (e.g. H4H2138P), 161 and 169 (e.g. H4H2140P), 177and 185 (e.g. H4H2143P), 193 and 201 (e.g. H4H2146P), 209 and 217 (e.g.H4H2147P), 225 and 233 (e.g. H4H2148P), 241 and 249 (e.g. H4H2151P), 257and 265 (e.g. H4H2153P), 273 and 281 (e.g. H4H2154P), 289 and 297 (e.g.H4H2290P), 305 and 313 (e.g. H1M1819N), 321 and 329 (e.g. H2M1821N), 337and 345 (e.g. H2M1824N), 353 and 361 (e.g. H2M1827N), 369 and 377 (e.g.H1M1828N), 385 and 393 (e.g. H2M1829N), 401 and 409 (e.g. H2M1930N), 417and 425 (e.g. H2M1934N), 433 and 441 (e.g. H2M1936N), 449 and 457 (e.g.H2M1937N), 465 and 473 (e.g. H2M1938N), or 481 and 489 (e.g. H1M1940N).

The present invention includes anti-ErbB3 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds ErbB3 and a pharmaceutically acceptable carrier. In arelated aspect, the invention features a composition which is acombination of an ErbB3 inhibitor and a second therapeutic agent. In oneembodiment, the ErbB3 inhibitor is an antibody or fragment thereof. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an ErbB3 inhibitor. Exemplary agents thatmay be advantageously combined with an ErbB3 inhibitor include, withoutlimitation, other agents that inhibit ErbB3 activity (including otherantibodies or antigen-binding fragments thereof, peptide inhibitors,small molecule antagonists, etc) and/or agents which interfere withErbB3 upstream or downstream signaling.

In yet another aspect, the invention provides methods for inhibitingErbB3 activity using an anti-ErbB3 antibody or antigen-binding portionof an antibody of the invention, wherein the therapeutic methodscomprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by removal, inhibition or reduction of ErbB3 activity. Theanti-ErbB3 antibody or antibody fragment of the invention may functionto block the interaction between ErbB3 and an ErbB3 binding partner(e.g., neuregulin-1), or otherwise inhibit the signaling activity ofErbB3.

The present invention also includes the use of an anti-ErbB3 antibody orantigen binding portion of an antibody of the invention in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by ErbB3 activity in a patient.

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

DEFINITIONS

The expressions “ErbB3” and “ErbB3 fragment,” as used herein refer tothe human ErbB3 protein or fragment unless specified as being from anon-human species (e.g., “mouse ErbB3,” “mouse ErbB3 fragment,” “monkeyErbB3,” “monkey ErbB3 fragment,” etc.). The extracellular domain ofhuman ErbB3 has the amino acid sequence shown in, e.g., amino acids1-613 of SEQ ID NOs:497-499.

The term “ErbB3 ligand,” as used herein, means a protein capable ofbinding to the extracellular domain of human ErbB3 protein to transmit abiological signal in vivo. The term “ErbB3 ligand” includes neuregulin-1(NRG1) and neuregulin-2 (NRG2).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-ErbB3 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3, (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3, (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” human ErbB3, as used in the context of the present invention,includes antibodies that bind human ErbB3 or portion thereof with aK_(D) of less than about 1000 nM, less than about 500 nM, less thanabout 300 nM, less than about 200 nM, less than about 100 nM, less thanabout 90 nM, less than about 80 nM, less than about 70 nM, less thanabout 60 nM, less than about 50 nM, less than about 40 nM, less thanabout 30 nM, less than about 20 nM, less than about 10 nM, less thanabout 5 nM, less than about 4 nM, less than about 3 nM, less than about2 nM, less than about 1 nM or less than about 0.5 nM, as measured in asurface plasmon resonance assay. (See, e.g., Example 3, herein). Anisolated antibody that specifically binds human ErbB3 may, however, havecross-reactivity to other antigens, such as ErbB3 molecules from other(non-human) species.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to ErbB3: (i) interferes with theinteraction between ErbB3 or an ErbB3 fragment and an ErbB3 ligand(e.g., neuregulin 1), and/or (ii) results in inhibition of at least onebiological function of ErbB3. The inhibition caused by an ErbB3neutralizing or blocking antibody need not be complete so long as it isdetectable using an appropriate assay. Exemplary assays for detectingErbB3 inhibition are described herein.

The anti-ErbB3 antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-ErbB3 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-ErbB3 antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Biological Characteristics of the Antibodies

The antibodies of the present invention block the interaction betweenErbB3 and its ligand neuregulin-1 (NRG1). As used herein, the expression“blocks the interaction between ErbB3 and NRG1” means that, in an assayin which the physical interaction between ErbB3 and NRG1 can be detectedand/or quantified, the addition of an antibody of the invention reducesthe interaction between ErbB3 and NRG1 by at least 50%. A non-limiting,exemplary assay that can be used to determine if an antibody blocks theinteraction between human ErbB3 and NRG1 is illustrated in Example 4,herein. In this Example, antibodies are mixed with ErbB3 protein, andthen the antibody/ErbB3 mixture is applied to a surface coated with NRG1protein. After washing away unbound molecules, the amount of ErbB3 boundto the NRG1-coated surface is measured. By using varying amounts ofantibody in this assay format, the amount of antibody required to block50% of ErbB3 binding to NRG1 can be calculated and expressed as an IC₅₀value. The present invention includes anti-ErbB3 antibodies that exhibitan IC₅₀ of less than about 600 pM when tested in an ErbB3/NRG1 bindingassay as described above, or a substantially similar assay. For example,the invention includes anti-ErbB3 antibodies that exhibit an IC₅₀ ofless than about 600, 500, 400, 300, 290, 280, 270, 260, 250, 240, 230,220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80,70, 60, 50, 40, 30, 20, 10, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 pM when tested in an ErbB3/NRG1 binding assay as described above,or a substantially similar assay.

Alternatively, one can determine whether an antibody blocks theinteraction between ErbB3 and NRG1 by using a cell-based assay formatthat detects changes in NRG1-induced cellular signaling. Exemplary assayformats of this type are illustrated in Examples 6 and 8, herein. Inthese Examples, the extent of phosphorylation of the kinase Akt and/orErbB3 in cells following treatment with NRG1 in the presence of varyingamounts of anti-ErbB3 antibody is measured. In these assay formats, thepercent inhibition of Akt and/or ErbB3 phosphorylation caused by thepresence of an anti-ErbB3 antibody serves as an indicator of the extentto which the antibody blocks the interaction between ErbB3 and NRG1. Thepresent invention includes antibodies that inhibit Akt or ErbB3phosphorylation by at least 60% when tested in an Akt or ErbB3phosphorylation assay as described above, or a substantially similarassay. For example, the invention includes anti-ErbB3 antibodies thatinhibit Akt or ErbB3 phosphorylation by at least about 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% when tested in an Akt or ErbB3phosphorylation assay as described above, or a substantially similarassay.

The anti-ErbB3 antibodies of the present invention also exhibit one ormore of the following properties: (1) the ability to induceinternalization of cell surface expressed ErbB3 (see, e.g., Example 5,herein); (2) the ability to inhibit NRG1-stimulated tumor cell growth invitro, either alone or in combination with EGFR inhibition (see, e.g.,Example 7, herein); and (3) the ability to inhibit tumor growth inanimals (see, e.g., Example 9, herein).

Epitope Mapping and Related Technologies

The ErbB3 protein, like all ErbB/HER family members, contains fourextracellular domains, referred to as “Domain I,” “Domain II,” “DomainIII,” and “Domain IV.” Domain I is the sequence of amino acidsrepresented by amino acids 1 through 190 of SEQ ID NO:498; Domain II isthe sequence of amino acids represented by amino acids 191 through 308of SEQ ID NO:498; Domain III is the sequence of amino acids representedby amino acids 309 through 499 of SEQ ID NO:498; and Domain IV is thesequence of amino acids represented by amino acids 500 through 624 ofSEQ ID NO:498.

The present invention includes anti-ErbB3 antibodies which interact withone or more epitopes found within Domain III of the extracellular domainof ErbB3. The epitope(s) may consist of one or more contiguous sequencesof 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) amino acids located within Domain III of ErbB3.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) located within Domain III ofErbB3. According to certain embodiments of the present invention,anti-ErbB3 antibodies are provided which interact with one or more aminoacids located within one or more Domain III amino acid segments selectedfrom the group consisting of amino acids 345-367 of SEQ ID NO:498, aminoacids 423-439 of SEQ ID NO:498; and amino acids 451-463 of SEQ IDNO:498. For example, the present invention includes anti-ErbB3antibodies which interact with at least one amino acid within each ofthe aforementioned Domain III amino acid segments (i.e., within each ofamino acids 345-367, 423-439, and 451-463 of SEQ ID NO:498).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. (See, e.g.,Example 11 herein). In general terms, the hydrogen/deuterium exchangemethod involves deuterium-labeling the protein of interest, followed bybinding the antibody to the deuterium-labeled protein. Next, theprotein/antibody complex is transferred to water to allowhydrogen-deuterium exchange to occur at all residues except for theresidues protected by the antibody (which remain deuterium-labeled).After dissociation of the antibody, the target protein is subjected toprotease cleavage and mass spectrometry analysis, thereby revealing thedeuterium-labeled residues which correspond to the specific amino acidswith which the antibody interacts. See, e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-ErbB3 antibodies that bindto the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g., H1M1819N, H2M1821N, H2M1824N, H2M1827N,H1M1828N, H2M1829N, H2M1930N, H2M1934N, H2M1938N, H1M1940N, etc.).Likewise, the present invention also includes anti-ErbB3 antibodies thatcompete for binding to ErbB3 or an ErbB3 fragment with any of thespecific exemplary antibodies described herein (e.g., H1M1819N,H2M1821N, H2M1824N, H2M1827N, H1M1828N, H2M1829N, H2M1930N, H2M1934N,H2M1938N, H1M1940N, etc.).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-ErbB3 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-ErbB3antibody of the invention, the reference antibody is allowed to bind toan ErbB3 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the ErbB3 molecule is assessed. Ifthe test antibody is able to bind to ErbB3 following saturation bindingwith the reference anti-ErbB3 antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-ErbB3antibody. On the other hand, if the test antibody is not able to bind tothe ErbB3 molecule following saturation binding with the referenceanti-ErbB3 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-ErbB3 antibody of theinvention. Additional routine experimentation (e.g., peptide mutationand binding analyses) can then be carried out to confirm whether theobserved lack of binding of the test antibody is in fact due to bindingto the same epitope as the reference antibody or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antibodies bind to the same (or overlapping)epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibodyinhibits binding of the other by at least 50% but preferably 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodiesare deemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies aredeemed to have “overlapping epitopes” if only a subset of the amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

To determine if an antibody competes for binding with a referenceanti-ErbB3 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to an ErbB3 molecule under saturatingconditions followed by assessment of binding of the test antibody to theErbB3 molecule. In a second orientation, the test antibody is allowed tobind to an ErbB3 molecule under saturating conditions followed byassessment of binding of the reference antibody to the ErbB3 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the ErbB3 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to ErbB3.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the same epitope as the reference antibody, but maysterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human ErbB3.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to ErbB3 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-ErbB3 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman ErbB3. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-ErbB3 antibody-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-ErbB3 antibody or antibody fragment that isessentially bioequivalent to an anti-ErbB3 antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-ErbB3 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-ErbB3 antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-ErbB3antibodies bind to human ErbB3 but not to ErbB3 from other species. Thepresent invention also includes anti-ErbB3 antibodies that bind to humanErbB3 and to ErbB3 from one or more non-human species. For example, theanti-ErbB3 antibodies of the invention may bind to human ErbB3 and maybind or not bind, as the case may be, to one or more of mouse, rat,guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow,horse, camel, cynomologous, marmoset, rhesus or chimpanzee ErbB3.

Immunoconjugates

The invention encompasses anti-ErbB3 monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-ErbB3 antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human ErbB3 or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target (e.g., EGFR,EGFRvIII, ErbB2/HER2, ErbB4, VEGF or Ang2) or is conjugated to atherapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82 I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-ErbB3 antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with ErbB3 activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering anti-ErbB3antibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by ErbB3 activity or treatable by blockingthe interaction between ErbB3 and an ErbB3 ligand (e.g., NRG1). Theantibodies and antigen-binding fragments of the present invention may beused to treat, e.g., primary and/or metastatic tumors arising in thebrain and meninges, oropharynx, lung and bronchial tree,gastrointestinal tract, male and female reproductive tract, muscle,bone, skin and appendages, connective tissue, spleen, immune system,blood forming cells and bone marrow, liver and urinary tract, andspecial sensory organs such as the eye. In certain embodiments, theantibodies and antigen-binding fragments of the invention are used totreat one or more of the following cancers: renal cell carcinoma,pancreatic carcinoma, breast cancer, head and neck cancer, prostatecancer, malignant gliomas, osteosarcoma, colorectal cancer, gastriccancer (e.g., gastric cancer with MET amplification), malignantmesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer,non-small cell lung cancer (e.g., EGFR-dependent non-small cell lungcancer), synovial sarcoma, thyroid cancer, or melanoma.

The present invention also provides methods for treating a tumor whichis resistant to, or has become resistant to anti-EGFR or anti-HER2therapy. For example, the present invention includes methods whichcomprise (a) identifying a patient having a tumor which is resistant, orhas become resistant, to one or more of an anti-EGFR antibody (e.g.,cetuximab), a small molecule inhibitor of EGFR (e.g., erlotinib), ananti-HER2 antibody (e.g. trastuzumab), and/or a small molecule inhibitorof HER2; and (b) administering to the patient an anti-ErbB3 antibody ofthe present invention, either alone, or in combination with an anti-EGFRantibody (e.g., cetuximab), a small molecule inhibitor of EGFR (e.g.,erlotinib), an anti-HER2 antibody (e.g. trastuzumab), and/or a smallmolecule inhibitor of HER2. Combination therapies are discussed in moredetail below.

Combination Therapies

The present invention includes therapeutic administration regimens whichcomprise administering an anti-ErbB3 antibody of the present inventionin combination with at least one additional therapeutically activecomponent. Non-limiting examples of such additional therapeuticallyactive components include other ErbB3 antagonists (e.g., a secondanti-ErbB3 antibody or small molecule inhibitor of ErbB3), an antagonistof ErbB2/HER2 (e.g., anti-HER2 antibody [e.g., trastuzumab] or smallmolecule inhibitor of HER2 activity), an antagonist of ErbB4 (e.g.,anti-ErbB4 antibody or small molecule inhibitor of ErbB4 activity), anantagonist of epidermal growth factor receptor (EGFR) (e.g., anti-EGFRantibody [e.g., cetuximab or panitumumab] or small molecule inhibitor ofEGFR activity [e.g., erlotinib or gefitinib]), an antagonist of EGFRvIII(e.g., an antibody that specifically binds EGFRvIII), a VEGF antagonist(e.g., a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,411 (also referred toherein as a “VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib), or an anti-DLL4 antibody (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), etc.).Other agents that may be beneficially administered in combination withthe anti-ErbB3 antibodies of the invention include cytokine inhibitors,including small-molecule cytokine inhibitors and antibodies that bind tocytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11,IL-12, IL-13, IL-17, IL-18, or to their respective receptors. Thepresent invention also includes therapeutic combinations comprising anyof the anti-ErbB3 antibodies mentioned herein and an inhibitor of one ormore of VEGF, DLL4, EGFR, or any of the aforementioned cytokines,wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme,an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fabfragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAbfragment; or other engineered molecules, such as diabodies, triabodies,tetrabodies, minibodies and minimal recognition units). The anti-ErbB3antibodies of the invention may also be administered in combination withantivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. Theanti-ErbB3 antibodies of the invention may also be administered as partof a treatment regimen that also includes radiation treatment and/orconventional chemotherapy. The additional therapeutically activecomponent(s) may be administered prior to, concurrent with, or after theadministration of an anti-ErbB3 antibody of the present invention; (forpurposes of the present disclosure, such administration regimens areconsidered the administration of an anti-ErbB3 antibody “in combinationwith” a therapeutically active component of the invention).

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-ErbB3 antibody may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an anti-ErbB3 antibody. As used herein, “sequentiallyadministering” means that each dose of anti-ErbB3 antibody isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of ananti-ErbB3 antibody, followed by one or more secondary doses of theanti-ErbB3 antibody, and optionally followed by one or more tertiarydoses of the anti-ErbB3 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-ErbB3 antibody.Thus, the “initial dose” is the dose which is administered at thebeginning of the treatment regimen (also referred to as the “baselinedose”); the “secondary doses” are the doses which are administered afterthe initial dose; and the “tertiary doses” are the doses which areadministered after the secondary doses. The initial, secondary, andtertiary doses may all contain the same amount of anti-ErbB3 antibody,but generally may differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount ofanti-ErbB3 antibody contained in the initial, secondary and/or tertiarydoses varies from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, two or more(e.g., 2, 3, 4, or 5) doses are administered at the beginning of thetreatment regimen as “loading doses” followed by subsequent doses thatare administered on a less frequent basis (e.g., “maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of anti-ErbB3 antibody which is administered to a patient priorto the administration of the very next dose in the sequence with nointervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-ErbB3 antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Any of the exemplary anti-ErbB3 antibodies disclosed herein may be usedin the context of the foregoing administration regimens.

Diagnostic Uses of the Antibodies

The anti-ErbB3 antibodies of the present invention may also be used todetect and/or measure ErbB3 in a sample, e.g., for diagnostic purposes.For example, an anti-ErbB3 antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofErbB3. Exemplary diagnostic assays for ErbB3 may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-ErbB3antibody of the invention, wherein the anti-ErbB3 antibody is labeledwith a detectable label or reporter molecule. Alternatively, anunlabeled anti-ErbB3 antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure ErbB3 in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in ErbB3 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of ErbB3 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of ErbB3 in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal ErbB3 levels or activity) will be measured to initiallyestablish a baseline, or standard, level of ErbB3. This baseline levelof ErbB3 can then be compared against the levels of ErbB3 measured insamples obtained from individuals suspected of having a ErbB3 relateddisease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human ErbB3

An immunogen comprising the ecto-domain of human ErbB3 was administereddirectly, with an adjuvant to stimulate the immune response, to aVELOCIMMUNE® mouse comprising DNA encoding human Immunoglobulin heavyand kappa light chain variable regions. The antibody immune response wasmonitored by a ErbB3-specific immunoassay. When a desired immuneresponse was achieved splenocytes were harvested and fused with mousemyeloma cells to preserve their viability and form hybridoma cell lines.The hybridoma cell lines were screened and selected to identify celllines that produce ErbB3-specific antibodies. Using this techniqueseveral anti-ErbB3 chimeric antibodies (i.e., antibodies possessinghuman variable domains and mouse constant domains) were obtained;exemplary antibodies generated in this manner were designated asfollows: H1M1819N, H2M1821N, H2M1824N, H2M1827N, H1M1828N, H2M1829N,H2M1930N, H2M1934N, H2M1936N, H2M1937N, H2M1938N, and H1M1940N.

Anti-ErbB3 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in US2007/0280945A1. Using this method, several fully human anti-ErbB3antibodies (i.e., antibodies possessing human variable domains and humanconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as follows: H4H2084P, H4H2092P, H4H2094P,H4H2098P, H4H2102P, H4H2108P, H4H2111P, H4H2114P, H4H2132P, H4H2138P,H4H2140P, H4H2143P, H4H2146P, H4H2147P, H4H2148P, H4H2151P, H4H2153P,H4H2154P, and H4H2290P.

Certain biological properties of the exemplary anti-ErbB3 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-ErbB3 antibodies and their correspondingantibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 2084P 2 4 6 8 10 12 14 16 2092P 18 20 22 24 26 28 3032 2094P 34 36 38 40 42 44 46 48 2098P 50 52 54 56 58 60 62 64 2102P 6668 70 72 74 76 78 80 2108P 82 84 86 88 90 92 94 96 2111P 98 100 102 104106 108 110 112 2114P 114 116 118 120 122 124 126 128 2132P 130 132 134136 138 140 142 144 2138P 146 148 150 152 154 156 158 160 2140P 162 164166 168 170 172 174 176 2143P 178 180 182 184 186 188 190 192 2146P 194196 198 200 202 204 206 208 2147P 210 212 214 216 218 220 222 224 2148P226 228 230 232 234 236 238 240 2151P 242 244 246 248 250 252 254 2562153P 258 260 262 264 266 268 270 272 2154P 274 276 278 280 282 284 286288 2290P 290 292 294 296 298 300 302 304 1819N 306 308 310 312 314 316318 320 1821N 322 324 326 328 330 332 334 336 1824N 338 340 342 344 346348 350 352 1827N 354 356 358 360 362 364 366 368 1828N 370 372 374 376378 380 382 384 1829N 386 388 390 392 394 396 398 400 1930N 402 404 406408 410 412 414 416 1934N 418 420 422 424 426 428 430 432 1936N 434 436438 440 442 444 446 448 1937N 450 452 454 456 458 460 462 464 1938N 466468 470 472 474 476 478 480 1940N 482 484 486 488 490 492 494 496

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H4H”, “H1M”, “H2M”), followed by anumerical identifier (e.g. “2084” as shown in Table 1), followed by a“P” or “N” suffix. Thus, according to this nomenclature, an antibody maybe referred to herein as, e.g., “H4H2084P”. The H4H, H1M, and H2Mprefixes on the antibody designations used herein indicate theparticular Fc region of the antibody. For example, an “H2M” antibody hasa mouse IgG2 Fc, whereas an “H4H” antibody has a human IgG4 Fc. As willbe appreciated by a person of ordinary skill in the art, an H1M or H2Mantibody can be converted to an H4H antibody, and vice versa, but in anyevent, the variable domains (including the CDRs)—which are indicated bythe numerical identifiers shown in Table 1—will remain the same. The Pand N suffixes on the antibody designations used herein refer toantibodies having heavy and light chains with identical CDR sequencesbut with sequence variations in regions that fall outside of the CDRsequences (i.e., in the framework regions). Thus, P and N variants of aparticular antibody have identical CDR sequences within their heavy andlight chain variable regions but differ from one another within theirframework regions.

Control Constructs Used in the Following Examples

Various control constructs (anti-ErbB3 antibodies) were included in thefollowing experiments for comparative purposes. The control constructsare designated as follows: Control I: a human anti-ErbB3 antibody withheavy and light chain variable domains having the amino acid sequencesof the corresponding domains of “Mab#6,” as set forth in U.S. Pat. No.7,846,440; Control II: a human anti-ErbB3 antibody with heavy and lightchain variable domains having the amino acid sequences of thecorresponding domains of “U1-59,” as set forth in U.S. Pat. No.7,705,130; and Control III: a human anti-ErbB3 antibody with heavy andlight chain variable domains having the amino acid sequences of thecorresponding domains of “U1-53,” as set forth in U.S. Pat. No.7,705,130.

Example 3 Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-ErbB3 Antibodies

Binding affinities and kinetic constants of human monoclonal anti-ErbB3antibodies were determined by surface plasmon resonance at 25° C. and37° C. (Tables 2-4). Measurements were conducted on a Biacore 2000 orT100 instrument. Antibodies, expressed with either mouse Fc (prefix H1M;H2M) or human IgG4 Fc (prefix H4H), were captured on an anti-mouse oranti human-Fc sensor surface (Mab capture format), and soluble monomeric(ErbB3.mmh; SEQ ID NO:497,) or dimeric (ErbB3-hFc; SEQ ID NO:498 orErbB3-mFc; SEQ ID NO:499) protein was injected over the surface. Kineticassociation (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d) I k_(a);and t_(1/2) (min)=(ln 2/(60*k_(d)). Several clones showed sub-nanomolaraffinity to monomeric (hErbB3.mmh) ErbB3 protein.

TABLE 2 Biacore Binding Affinities of Hybridoma mAbs (H1M and H2M) at25° C. Binding at 25° C./Mab Capture Format Antibody Analyte ka (Ms⁻¹)kd (s⁻¹) K_(D) (Molar) T½ H1M1819N hErbB3.mmh 3.19E+05 3.22E−04 1.01E−0936 hErbB3.hFc 4.80E+05 5.88E−05 1.22E−10 196 H2M1821N hErbB3.mmh2.29E+05 1.99E−04 8.67E−10 58 hErbB3.hFc 4.61E+05 1.90E−05 4.13E−11 608H2M1824N hErbB3.mmh 2.23E+05 4.56E−05 2.05E−10 254 hErbB3.hFc 4.31E+051.44E−06 3.34E−12 8026 H2M1827N hErbB3.mmh 2.19E+05 8.96E−05 4.09E−10129 hErbB3.hFc 4.39E+05 7.58E−06 1.73E−11 1524 H1M1828N hErbB3.mmh5.13E+05 2.65E−04 5.15E−10 44 hErbB3.hFc 1.56E+06 4.34E−05 2.79E−11 266H2M1829N hErbB3.mmh 2.30E+05 6.46E−05 2.81E−10 179 hErbB3.hFc 4.36E+058.61E−06 1.98E−11 1341 H2M1930N hErbB3.mmh 1.96E+05 1.09E−04 5.59E−10106 hErbB3.hFc 4.04E+05 8.27E−06 2.05E−11 1396 H2M1934N hErbB3.mmh1.68E+05 7.35E−05 4.38E−10 157 hErbB3.hFc 3.59E+05 7.97E−06 2.22E−111450 H2M1936N hErbB3.mmh 4.32E+04 2.85E−04 6.59E−09 41 hErbB3.hFc6.41E+04 6.97E−05 1.09E−09 166 H2M1937N hErbB3.mmh 8.26E+04 5.63E−056.82E−10 205 hErbB3.hFc 1.10E+05 1.27E−05 1.16E−10 908 H2M1938NhErbB3.mmh 2.41E+05 1.44E−04 5.99E−10 80 hErbB3.hFc 4.51E+05 1.36E−053.01E−11 851 H1M1940N hErbB3.mmh 2.89E+05 1.38E−04 4.77E−10 84hErbB3.hFc 4.60E+05 2.43E−05 5.29E−11 475 Control I hErbB3.mmh 5.14E+042.15E−04 4.18E−09 54 hErbB3.hFc 4.63E+04 1.63E−05 3.51E−10 711 ControlII hErbB3.mmh 1.41E+05 3.24E−04 2.30E−09 36 hErbB3.hFc 1.53E+05 4.28E−052.80E−10 270 Control III hErbB3.mmh 1.54E+06 3.15E−02 2.05E−08 0.4hErbB3.hFc 3.78E+06 8.84E−05 2.34E−11 131

TABLE 3 Biacore Binding Affinities of Human Fc mAbs (H4H) at 25° C.Binding at 25° C./Mab Capture Format Analyte ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) T½ H4H1819N hErbB3.mmh 8.10E+06 3.51E−04 4.35E−11 33 hErbB3.mFc1.64E+07 1.54E−05 9.43E−13 748 H4H1821N hErbB3.mmh 3.80E+06 1.92E−045.10E−11 60 hErbB3.mFc 1.22E+07 4.33E−06 3.55E−13 2665 H4H2084PhErbB3.mmh 2.49E+06 5.96E−05 2.39E−11 179 hErbB3.mFc 3.83E+06 3.95E−061.03E−12 2695 H4H2092P hErbB3.mmh 3.72E+06 1.03E−04 2.78E−11 103hErbB3.mFc 5.16E+06 7.46E−06 1.45E−12 1429 H4H2094P hErbB3.mmh 1.89E+061.37E−05 7.22E−12 780 hErbB3.mFc 3.03E+06 1.37E−06 4.52E−13 7779H4H2098P hErbB3.mmh 1.14E+06 7.89E−05 6.90E−11 135 hErbB3.mFc 2.21E+068.97E−06 4.06E−12 1188 H4H2102P hErbB3.mmh 8.86E+05 4.88E−05 5.51E−11218 hErbB3.mFc 1.58E+06 2.26E−06 1.43E−12 4721 H4H2108P hErbB3.mmh1.95E+06 8.13E−05 4.18E−11 131 hErbB3.mFc 2.96E+06 4.33E−06 1.46E−122458 H4H2111P hErbB3.mmh 2.21E+06 1.18E−04 5.31E−11 91 hErbB3.mFc3.50E+06 8.69E−06 2.49E−12 1225 H4H2114P hErbB3.mmh 9.29E+05 9.88E−051.06E−10 108 hErbB3.mFc 1.48E+06 7.98E−06 5.41E−12 1335 H4H2132PhErbB3.mmh 2.16E+06 3.81E−05 1.77E−11 279 hErbB3.mFc 3.49E+06 3.35E−069.58E−13 3183 H4H2138P hErbB3.mmh 2.39E+06 5.01E−05 2.09E−11 213hErbB3.mFc 3.71E+06 5.46E−06 1.47E−12 1952 H4H2140P hErbB3.mmh 1.66E+063.27E−05 1.98E−11 326 hErbB3.mFc 2.51E+06 9.86E−07 3.93E−13 10797H4H2143P hErbB3.mmh 1.83E+06 9.73E−05 5.31E−11 109 hErbB3.mFc 2.86E+064.63E−06 1.75E−12 2302 H4H2146P hErbB3.mmh 2.79E+06 3.46E−05 1.24E−11308 hErbB3.mFc 4.54E+06 1.98E−06 4.35E−13 5392 H4H2147P hErbB3.mmh2.47E+06 1.08E−04 4.38E−11 98 hErbB3.mFc 3.33E+06 4.58E−06 1.50E−12 2325H4H2148P hErbB3.mmh 3.98E+06 3.47E−05 8.71E−12 307 hErbB3.mFc 5.91E+061.74E−06 2.95E−13 6110 H4H2151P hErbB3.mmh 3.04E+06 2.86E−05 9.42E−12372 hErbB3.mFc 4.48E+06 9.52E−07 2.13E−13 11186 H4H2153P hErbB3.mmh2.94E+06 3.43E−05 1.17E−11 311 hErbB3.mFc 3.67E+06 1.24E−06 3.37E−138603 H4H2154P hErbB3.mmh 2.13E+06 3.73E−05 1.76E−11 285 hErbB3.mFc3.25E+06 9.77E−07 3.00E−13 10901 H4H2290P hErbB3.mmh 5.82E+05 6.72E−051.15E−10 159 hErbB3.mFc 8.00E+05 1.13E−05 1.40E−11 945

TABLE 4 Biacore Binding Affinities of Select mAbs at 37° C. Binding at37° C./Mab Capture Format K_(D) Analyte ka (Ms⁻¹) kd (s⁻¹) (Molar) T½H4H1819N hErbB3.mmh 1.21E+07 1.56E−03 1.29E−10 7 hErbB3.mFc 3.68E+075.62E−05 1.53E−12 206 H4H1821N hErbB3.mmh 6.49E+06 1.08E−03 1.67E−10 11hErbB3.mFc 1.87E+07 3.55E−05 1.89E−12 326 Control I hErbB3.mmh 1.58E+055.48E−04 3.47E−09 21 hErbB3.mFc 2.60E+05 1.01E−04 3.90E−10 114 ControlII hErbB3.mmh 3.23E+05 1.34E−03 4.13E−09 9 hErbB3.mFc 1.44E+06 1.37E−049.50E−11 84 Control III hErbB3.mmh 3.40E+06 7.90E−02 2.40E−08 0.1hErbB3.mFc 1.05E+07 1.77E−04 1.68E−11 65

As shown in Tables 2-4, many of the exemplary antibodies tested in thisExample exhibited high affinity binding to ErbB3 that was superior orequivalent to the binding affinities of the control antibodies tested.Of note, several of the anti-ErbB3 antibodies of the present inventionexhibited sub-nanomolar affinity to monomeric (hErbB3.mmh) ErbB3protein.

Example 4 Anti-ErbB3 Antibodies Block Neuregulin 1b Binding to HumanErbB3

To further characterize anti-hErbB3 mAbs of the present invention, theirability to block ligand binding was examined via ELISA. Plates werecoated with neuregulin1b (1 μg/ml) overnight and then antibodies (0-50nM) were incubated (1 hr, 25° C.) with either 50 μM ErbB3-hFc (SEQ IDNO:498; for hybridomas) or 50 μM ErbB3-mFc (SEQ ID NO:499; for hlgG4antibodies) and then added to coated plates and allowed to incubate foran additional 1 hr at 25° C. Plates were washed and non-sequestered(plate bound) ErbB3 was detected with an anti-Fc poly conjugated withhorseradish peroxidase (HRP). Plates were developed with TMB(3,3′,5,5′-tetramethylbenzidine) and neutralized with sulfuric acidbefore reading absorbance at 450 nm on a Victor X5 plate reader. Dataanalysis used a sigmoidal dose-response model within Prism™ software.The calculated IC₅₀ value, defined as 50% of the antibody concentrationrequired to achieve maximum blocking, was used as an indicator ofblocking potency. Maximal blocking for each antibody was calculated bytaking the difference in absorbance from zero to 50 nM antibody on theinhibition curve, divided by the difference in absorbance from 50 pM tozero ErbB3 on the dose curve. Results are shown in Tables 5 and 6.

TABLE 5 IC₅₀ Values for Anti-ErbB3 Hybridoma mAbs (H1M and H2M) MaximalBlocking Antibody IC50 (M) (%) H1M1819N 3.15E−11 92 H2M1821N 2.85E−11 96H2M1824N 2.51E−11 99 H2M1827N 2.29E−11 98 H1M1828N 3.00E−11 95 H2M1829N2.38E−11 98 H2M1930N 2.22E−11 87 H2M1934N 2.61E−11 80 H2M1936N 5.27E−1091 H2M1937N 4.40E−11 95 H2M1938N 2.49E−11 85 H1M1940N 5.30E−10 80

TABLE 6 IC₅₀ Values for Anti-ErbB3 Human Fc mAbs (H4H) Maximal BlockingAntibody IC50 (M) (%) H4H1819N 2.00E−11 99 H4H1821N 1.80E−11 99 H4H2084P8.73E−11 95 H4H2092P 5.94E−11 100 H4H2094P 9.00E−11 92 H4H2098P 1.35E−1095 H4H2102P 1.86E−10 90 H4H2108P 1.16E−10 91 H4H2111P 4.97E−11 92H4H2114P 1.63E−10 91 H4H2132P 9.57E−11 94 H4H2138P 1.06E−10 96 H4H2140P9.46E−11 83 H4H2143P 8.35E−11 92 H4H2146P 1.77E−10 83 H4H2147P 5.06E−1199 H4H2148P 5.08E−11 100 H4H2151P 7.51E−11 85 H4H2153P 7.40E−11 82H4H2154P 9.01E−11 91 H4H2290P 6.64E−11 99 Control I 5.74E−10 98 ControlIII 8.32E−11 96

As shown in Tables 5 and 6, several anti-ErbB3 antibodies of the presentinvention showed potent blocking and had IC₅₀ values that were at thetheoretical bottom (25 pM) of the assay.

Example 5 Anti-ErbB3 mAbs Effectively Internalize Cell Surface ErbB3

To determine if anti-ErbB3 mAbs effectively internalize cell surfacebound ErbB3, MCF-7 cells were incubated with select anti-ErbB3 antibody(10 μg/ml) for 30 minutes on ice, washed and then stained with a Dylight488 conjugated anti-human Fab (10 μg/ml) for 30 minutes on ice. Tubeswere then washed and split between an on ice and 37° C. incubation for 1hour. After incubation all tubes were placed on ice and a Dylight 488quenching antibody (50 μg/ml) was added. Solutions were incubated anadditional 1 hour on ice. Fluorescent signals were measured using anAccuri flow cytometer.

As a relative measure of the amount of ErbB3 that was internalized uponantibody binding and subsequent incubation at 37° C., the internalizedmean fluorescent intensity (MFI) was calculated as follows:

Internalized MFI=Total MFI−Surface MFI

where:

Surface MFI=(Total MFI−MFI of Quenched Sample)/QE

and

QE=1−[MFI of quenched sample at 4° C./MFI of unquenched sample at 4° C.]

The calculation of QE (quenching efficiency) assumes that nointernalization occurs at 4° C. Table 7 shows that all antibodies testedinduce HER3 internalization.

TABLE 7 Antibody-Induced HER3 Internalization at 37° C. Total MFISurface MFI Internalized MFI % Internalized Antibody mean ± S.D. mean ±S.D. mean ± S.D. mean ± S.D. H4H1819N 39233 ± 9261 22663 ± 5026 16570 ±5329 42 ± 7  H4H1821N 32351 ± 5658 11607 ± 4781 20744 ± 4993 64 ± 14Control I 19004 ± 5903 11598 ± 6602  7406 ± 1776 42 ± 20 Control II41517 ± 5696  23540 ± 11994 17977 ± 6299 45 ± 21 Control III 27349 ±5310 8010 ± 729 19338 ± 5934 69 ± 8 

Example 6 Inhibition of NRG1-Dependent Akt Phosphorylation by Anti-ErbB3Antibodies

Anti-ErbB3 antibodies were tested for their ability to inhibitphosphorylation of Akt in DU145 prostate cancer cells. Binding of NRG1to ErbB3 results in ErbB3 phosphorylation, which leads to recruitmentand activation of phosphatidylinositol 3-kinase (Pl3-K). Activated Pl3-Kphosphorylates and activates the kinase Akt. Thus, Akt phosphorylationis a downstream marker of ErbB3 receptor activation. DU145 cells wereseeded in 96-well plates and then serum-starved in medium containing 1%FBS overnight. Cells were then stimulated with 0.5 nM NRG1 (R&D Systems)for 30 min in the presence of 0.5 μg/ml human Fc control protein orvarious anti-ErbB3 antibodies at concentrations of 0.01, 0.1 or 0.5μg/ml. Each concentration of antibody was tested in triplicate. Cellswere lysed and the relative levels of phosphorylated Akt were determinedusing a phospho-Akt cell-based ELISA kit (R&D Systems), according to themanufacturer's instructions. The average percent inhibition of Aktphosphorylation for each anti-ErbB3 antibody versus the Fc control groupis shown in Table 8.

TABLE 8 Inhibition of Akt phosphorylation by Anti-ErbB3 AntibodiesPercent inhibition of Akt phosphorylation 0.01 μg/ml ErbB3 0.1 μg/mlErbB3 0.5 μg/ml ErbB3 Antibody antibody antibody antibody H1M1819N 29 7275 H2M1821N 11 68 73 H2M1824N 25 63 74 H2M1827N 16 73 75 H1M1828N 22 6675 H2M1829N 22 74 74 H2M1930N 39 64 66 H2M1934N 20 56 67 H2M1936N 6 3067 H2M1937N −13 41 71 H2M1938N 40 63 74 H1M1940N 13 50 68 Control I −146 60 Control II 4 7 51 Control III 3 45 55

This example illustrates that several of the anti-ErbB3 antibodies ofthe present invention exhibited more potent blockade of Aktphosphorylation than the control anti-ErbB3 antibodies. For example,antibodies H1M1819N, H2M1821N, H2M1824N, H2M1827N, H1M1828N, H2M1829N,H2M1930N and H2M1938N inhibited Akt phosphorylation by at least 60% atthe 0.1 μg/m/dose, while none of the control ErbB3 antibodies achievedan inhibition greater than 46% at that dose.

Example 7 Inhibition of Tumor Cell Growth by Anti-ErbB3 Antibodies

Select anti-ErbB3 antibodies were tested for their ability to inhibitthe growth of A431 epidermoid carcinoma cells when used in combinationwith EGFR blockade. A431 cells in 96-well plates were incubated inmedium containing 0.5% FBS and stimulated with 1 nM neuregulin-1 (NRG1)in the presence of an anti-EGFR antibody (1 μg/ml), an anti-ErbB3antibody (1 μg/ml) or anti-EGFR (1 μg/ml) plus anti-ErbB3 antibody at0.05, 0.25 or 1.0 μg/ml. The ligand (NRG1) and blocking antibodies (EGFR& ErbB3) were added at 0, 24 and 48 hrs during the 72-hour experiment.The relative change in the number of viable cells from the start oftreatment until the 72-hour time point was reached was determined usingstandard methods (Cell Proliferation Assay Kit; Promega). The averagepercent decrease in cell growth for each anti-ErbB3 antibody versus anisotype control group is shown in Table 9.

TABLE 9 Inhibition of A431 Cell Growth by Anti-ErbB3 Antibodies Percentdecrease in the growth of A431 cells anti-EGFR anti-EGFR anti-EGFRantibody + antibody + antibody + 1.0 μg/ml 0.05 μg/ml 0.25 μg/ml 1.0μg/ml ErbB3 ErbB3 ErbB3 ErbB3 Antibody antibody antibody antibodyantibody H1M1819N 34 61 78 91 H2M1821N 16 35 62 85 H2M1824N 33 45 68 85H2M1827N 15 53 75 84 H1M1828N 30 55 73 85 H2M1829N 31 53 76 89 H2M1930N3 23 36 39 H2M1934N −3 24 30 28 H2M1938N 5 37 56 60 H1M1940N −4 19 20 19Control I 19 31 37 56 Control III 7 22 21 32

This example illustrates that several of the anti-ErbB3 antibodies ofthe present invention exhibited more potent inhibition of A431 cellgrowth than the control anti-ErbB3 antibodies. For example, antibodiesH1M1819N, H2M1821N, H2M1824N, H2M1827N, H1M1828N and H2M1829N inhibitedcell growth by at least 60% at the 0.25 μg/m/dose when combined withanti-EGFR antibody, while control antibodies I and III inhibited cellgrowth by only 37% and 21%, respectively, under these experimentalconditions.

Example 8 Inhibition of ErbB3 and Akt Phosphorylation by Anti-ErbB3Antibodies

Select anti-ErbB3 antibodies were tested for their ability to inhibitNRG1-stimulated phosphorylation of ErbB3 and Akt in A431 epidermoidcarcinoma and MCF7 breast cancer cells. In the first experiment, A431cells were seeded in 6-well plates and incubated in complete mediumovernight. Cells were then serum-starved (0.5% FBS) for 1 hr andstimulated with 1 nM NRG1 (R&D Systems) for 30 min in the presence of5.0 μg/ml of isotype control or anti-ErbB3 antibody at 0.05, 0.25 or 5.0μg/ml. Cells were lysed and Western blots were performed usingantibodies against ErbB3 and Akt as well as their phosphorylated formsusing standard methods. The ratios of phosphorylated ErbB3 or Akt tototal ErbB3 or Akt were calculated and used to determine the percentinhibition of Akt or ErbB3 phosphorylation for each of the anti-ErbB3antibodies relative to isotype control. Results for the inhibition ofErbB3 or AKT phosphorylation in A431 cells are shown in Tables 10 and11, respectively.

TABLE 10 Percent Inhibition of ErbB3 Phosphorylation in A431 CellsAntibody 0.05 μg/ml 0.25 μg/ml 5.0 μg/ml H4H1819N 98 105 107 H4H1821N 63113 120 Control I −11 14 72 Control III −30 −9 −57

TABLE 11 Percent Inhibition of Akt Phosphorylation in A431 CellsAntibody 0.05 μg/ml 0.25 μg/ml 5.0 μg/ml H4H1819N 84 104 113 H4H1821N 67106 117 Control I 32 47 75 Control III 43 51 58

In a similar experiment, MCF7 cells were seeded in 6-well plates andincubated in complete medium for 2 days. Cells were then serum-starved(0.5% FBS) for 1 hr and then stimulated with 1 nM NRG1 (R&D Systems) for30 min in the presence of 5.0 μg/ml of isotype control or anti-ErbB3antibody at of 0.05, 0.25, 1.0 or 5.0 μg/ml. Western blots and dataanalysis were carried out in a similar manner for those experimentsconducted with A431 cells. Results for the inhibition of ErbB3 or AKTphosphorylation in MCF7 cells are shown in Tables 12 and 13,respectively.

TABLE 12 Percent Inhibition of ErbB3 Phosphorylation in MCF7 CellsAntibody 0.05 μg/ml 0.25 μg/ml 1.0 μg/ml 5.0 μg/ml H4H1819N 37 79 92 96H4H1821N 57 92 96 97 Control I 0 17 44 81 Control III 4 12 60 61

TABLE 13 Percent Inhibition of Akt Phosphorylation in MCF7 CellsAntibody 0.05 μg/ml 0.25 μg/ml 1.0 μg/ml 5.0 μg/ml H4H1819N 13 36 80 90H4H1821N 17 69 82 89 Control I 21 29 34 46 Control III 35 28 29 35

This Example illustrates that representative anti-ErbB3 antibodies ofthe invention exhibited superior ability to inhibit phosphorylation ofErbB3 and Akt compared to control antibodies under most of theexperimental conditions tested. In A431 cells, for example, H4H1819N andH4H1821N completely inhibited both ErbB3 and Akt phosphorylation at 0.25μg/ml while the control antibodies never achieved complete inhibition,even at 5.0 μg/ml. Similarly, in MCF7 cells, H4H1819N and H4H1821N at1.0 μg/ml inhibited ErbB3 and Akt phosphorylation to a greater extentthan either of the control anti-ErbB3 antibodies even at 5.0 μg/ml.

Example 9 Inhibition of Tumor Growth by Anti-ErbB3 Antibodies

Select anti-ErbB3 antibodies were tested for their ability to inhibitthe growth of human tumor xenografts in immunocompromised mice. Briefly,1-5×10⁶ A431 human epidermoid carcinoma cells or A549 human non-smallcell lung cancer cells (ATCC) were implanted subcutaneously into theflank of 6-8 week old SCID mice (Taconic, Hudson, N.Y.). After tumorsreached an average volume of 100-150 mm³, mice were randomized intogroups for treatment (n=6 mice per group). In the first experiment, micebearing A431 tumors were administered human Fc (SEQ ID NO:500, 12.5mg/kg), or anti-ErbB3 antibody (0.5 or 12.5 mg/kg). All antibodies wereadministered via subcutaneous injection twice per week for approximately3 weeks. Tumor volumes were measured twice per week over the course ofthe experiment and tumor weights were determined upon excision of tumorsat the conclusion of the experiment. Average tumor size relative to theFc treated group as well as final tumor weights were calculated for eachgroup. Results are summarized in Table 14.

TABLE 14 Inhibition of A431 Tumor Growth in SCID Mice (Hybridoma mAbs -H1M and H2M) Tumor Growth in mm³ from Start of Average % Average %Antibody Treatment Decrease in Avg Tumor Decrease in (mg/kg) (mean ±S.D.) Tumor Growth Weight (g) Tumor Weight hFc ctrl (12.5) 860 ± 358 —0.778 ± 0.268 — H1M1819N (0.5) 355 ± 178 59 0.520 ± 0.155 33 H2M1821N(0.5) 280 ± 131 67 0.428 ± 0.209 45 H2M1827N (0.5) 246 ± 71  71 0.432 ±0.152 45 H2M1829N (0.5) 392 ± 265 54 0.480 ± 0.283 38 Control I (0.5)862 ± 199  0 0.815 ± 0.190 −5 Control I (12.5) 299 ± 139 65 0.438 ±0.217 44

In a similar experiment, using selected antibodies in the human IgG4format yield similar results, as summarized in Table 15.

TABLE 15 Inhibition of A431 Tumor Growth in SCID Mice (Human Fc mAbs -H4H) Tumor Growth in mm³ from Start Average % Average % of TreatmentDecrease in Avg Tumor Decrease in Antibody (mg/kg) (mean ± S.D.) TumorGrowth Weight (g) Tumor Weight hFc ctrl (12.5) 797 ± 65 —  1.31 ± 0.142— H4H1819N (0.5) 161 ± 69 80 0.453 ± 0.010 65 H4H1819N (12.5) 110 ± 4786 0.458 ± 0.108 65 H4H1821N (0.5) 148 ± 73 81 0.482 ± 0.058 63 H4H1821N(12.5)  74 ± 100 91 0.392 ± 0.117 70 Control I (0.5)  675 ± 228 15 0.928± 0.215 29 Control I (12.5)  95 ± 51 88 0.361 ± 0.063 72 Control III(0.5)  409 ± 254 49 0.687 ± 0.269 48 Control III (12.5)  219 ± 129 730.545 ± 0.096 58

In similar experiments, the effect of various anti-ErbB3 antibodies onthe growth of A549 tumor xenografts was determined, as summarized inTable 16.

TABLE 16 Inhibition of A549 Tumor Growth in SCID Mice Tumor Growth inmm³ from Start of Average % Average % Antibody Treatment Decrease in AvgTumor Decrease in (mg/kg) (mean ± S.D.) Tumor Growth Weight (g) TumorWeight hFc ctrl (12.5) 727 ± 184 —  1.27 ± 0.332 — H4H1821N (0.2) 366 ±90  50 0.811 ± 0.145 37 H4H1821N (0.5) 347 ± 52  52 0.820 ± 0.245 36H4H1821N (2.5) 346 ± 106 52 0.783 ± 0.175 39 Control I (0.5) 719 ± 230 11.328 ± 0.363 −3.78 Control I (2.5) 614 ± 177 15 0.985 ± 0.198 23

As shown in this Example, antibodies H4H1819N and H4H1821N eachsignificantly inhibited tumor growth in vivo to an extent that wassuperior, or equivalent to the extent of tumor growth inhibitionobserved with administration of the control anti-ErbB3 antibodiestested.

Example 10 Inhibition of Tumor Growth by Anti-ErbB3 Antibodies inCombination with Agents that Block other ErbB Family Members

The effect of a combination treatment with H4H1821N plus an inhibitoryanti-EGFR antibody (“anti-EGFR mAb”) on human tumor xenograft growth wastested. Briefly, 2×10⁶ FaDu human hypopharyngeal carcinoma cells (ATCC)were implanted subcutaneously into the flank of 6-8 week old SCID mice(Taconic, Hudson, N.Y.). After tumors reached an average volume of150-200 mm³, mice were randomized into groups for treatment (n=6 miceper group). Mice were administered human Fc control protein (12.5mg/kg), H4H1821N (2.5 mg/kg), anti-EGFR mAb (10 mg/kg) or thecombination of H4H1821N plus anti-EGFR mAb (2.5+10 mg/kg). Allantibodies were administered via subcutaneous injection twice per week.Tumor volumes were measured twice per week over the course of theexperiment and tumor weights were determined upon excision of tumors atthe conclusion of the experiment. Averages (mean+/−standard deviation)of the tumor growth from the start of treatment and the tumor weightswere calculated for each treatment group. The percent decrease of tumorgrowth and tumor weight was calculated from comparison to the Fc controlgroup. The results are shown in Table 17.

TABLE 17 Inhibition of FaDu tumor xenograft growth in SCID mice TumorGrowth in mm³ from Start of Average % Average % Treatment Decrease inAverage Tumor Decrease in Antibody (mg/kg) (mean ± S.D.) Tumor GrowthWeight (g) Tumor Weight hFc control (12.5) 1099 ± 186  — 0.993 ± 0.176 —H4H1821N (2.5) 284 ± 175 74 0.522 ± 0.177 47 anti-EGFR mAb (10)  55 ±115 95 0.215 ± 0.120 78 H4H1821N + anti- −199 ± 38  118  0.024 ± 0.02098 EGFR mAb (2.5 + 10)

In a similar experiment, the effect of a combination treatment withH4H1821N plus the inhibitory anti-HER2 antibody clone 4D5v8 as describedin Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992) onhuman tumor xenograft growth was tested. Briefly, 1×10⁷ BT474 humanbreast cancer cells (ATCC) were implanted subcutaneously into the flankof 6-8 week old NCR nude mice (Taconic, Hudson, N.Y.). After tumorsreached an average volume of 150-200 mm³, mice were randomized intogroups for treatment (n=5 mice per group). Mice were administered humanFc control protein (25 mg/kg), H4H1821N (12.5 mg/kg), 4D5v8 (12.5 mg/kg)or the combination of H4H1821N plus 4D5v8 (12.5+12.5 mg/kg). Allantibodies were administered via subcutaneous injection twice per week.Tumor volumes were measured twice per week over the course of theexperiment. The average (mean+/−standard deviation) tumor growth fromthe start of treatment was calculated for each treatment group. Thepercent decrease of tumor growth was calculated from comparison to theFc control group. The results are shown in Table 18.

TABLE 18 Inhibition of BT474 tumor xenograft growth in nude mice Tumorgrowth in mm3 from start of treatment Average % Decrease in Antibody(mg/kg) (mean ± SD) Tumor Growth hFc control (25) 194 ± 39 — H4H1821N(12.5) 137 ± 65 29 4D5v8 (12.5)  34 ± 121 82 H4H1821N + 4D5v8 (12.5 +12.5) −79 ± 39 141 

This example illustrates that combined treatment with H4H1821N plusanti-EGFR or anti-HER2 antibodies provides more potent inhibition oftumor growth than the single agent treatments. In both FaDu and BT474tumor xenografts, the combination treatments, but not the single agents,caused the average tumor size to decrease (tumor regression).

Example 11 Epitope Mapping of H4H1821N Binding to ErbB3 by H/D Exchange

Experiments were conducted to determine the amino acid residues of ErbB3with which H4H1821N interacts. For this purpose H/D exchange epitopemapping was carried out. A general description of the H/D exchangemethod is set forth in e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256 A-265A.

To map the binding epitope(s) of antibody H4H1821N on ErbB3 via H/Dexchange, a recombinant construct consisting of the extracellular domainof hErbB3 (amino acids 1-613 of SEQ ID NO:498) with a C-terminalmyc-myc-hexahistidine tag (“hErbB3-mmH”) was used. hErbB3-mmH was firstdeglycosylated with PNGase F (New England BioLabs) under nativeconditions. Antibody H4H1821N was covalently attached toN-hydroxysuccinimide (NHS) agarose beads (GE Lifescience).

In the ‘on-solution/off-beads’ experiment (on-exchange in solutionfollowed by off-exchange on beads), the ligand (deglycosyatedhErbB3-mmH) was deuterated for 5 min or 10 min in PBS buffer preparedwith D₂O, and then bound to H4H1821N beads through a 2 min incubation.The ErbB3-bound beads were washed with PBS aqueous buffer (prepared withH₂O) and incubated for half of the on-exchange time in PBS buffer. Afterthe off-exchange, the bound ErbB3 was eluted from beads with an ice-coldlow pH TFA solution. The eluted ErbB3 was then digested with immobilizedpepsin (Thermo Scientific) for 5 min. The resulting peptides weredesalted using ZipTip® chromatographic pipette tips and immediatelyanalyzed by UltrafleXtreme matrix assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry (MS).

In the ‘on-beads/off-beads’ experiment (on-exchange on beads followed byoff-exchange on beads), ErbB3 was first bound to H4H1821N beads and thenincubated for 5 min or 10 min in D₂O for on-exchange. The followingsteps (off-exchange, pepsin digestion, and MS analysis) were carried outas described for the ‘on-solution/off-beads’ procedure. The centroidvalues or average mass-to-charge ratios (m/z) of all the detectedpeptides were calculated and compared between these two sets ofexperiments.

The results are summarized in Table 19 which provides a comparison ofthe centroid m/z values for all the detected peptides identified byliquid chromatography-matrix assisted laser desorption ionization(LC-MALDI) MS following the H/D exchange and peptic digest procedure.While the majority of the observed peptic peptides gave similar centroidvalues for both the on-solution/off-beads and on-beads/off-beadsexperiments, three segments corresponding to residues 345-367, 423-439,and 451-463 of the extracellular domain of ErbB3 (SEQ ID NO:498) haddelta centroid values (Δ) greater than or equal to 0.20 m/z in both the‘5 min on-/2.5 min off-exchange’ experiment (Experiment I) and the ‘10min on-/5 min off-exchange’ experiment (Experiment II). For purposes ofthe present Example, a positive difference (Δ) of at least 0.20 m/z inboth experiments indicates amino acids protected by antibody binding.Segments meeting this criterion are indicated by bold text and anasterisk (*) in Table 19.

TABLE 19 H4H1821N Binding to hErbB3-mmH Experiment I Experiment IIResidues 5 min on-/2.5 min off-exchange 10 min on-/5 min off-exchange(of SEQ ID On-solution/ On-Beads/ On-solution/ On-Beads/ NO: 498) OffBeads Off-Beads Δ Off Beads Off-Beads Δ 46-57 1287.52 1287.41 0.111287.58 1287.64 −0.06 58-63 844.97 844.97 0.00 845.04 844.99 0.06 58-661102.34 1102.25 0.09 1102.25 1102.30 −0.05 58-67 1265.65 1265.60 0.051265.57 1265.50 0.07 58-69 1477.84 1477.77 0.07 1477.79 1477.79 0.0159-69 1364.46 1364.48 −0.02 1364.39 1364.42 −0.03 61-69 1050.70 1050.670.03 1050.75 1050.68 0.07 75-96 2509.35 2509.27 0.08 2509.21 2509.210.01 76-96 2362.11 2362.09 0.01 2362.11 2361.97 0.14 84-96 1526.221526.05 0.16 1526.09 1526.08 0.01 84-98 1710.14 1710.11 0.03 1710.171710.07 0.11 84-99 1857.37 1857.34 0.03 1857.39 1857.33 0.06 86-961270.52 1270.50 0.02 1270.50 1270.45 0.05 100-114 1750.75 1750.60 0.151750.85 1750.75 0.11 100-114 1766.52 1766.55 −0.03 1766.63 1766.47 0.16100-120 2476.03 2475.80 0.23 2476.04 2475.96 0.08 103-117 1789.631789.48 0.14 1789.69 1789.44 0.24 112-120 1142.00 1141.95 0.05 1142.011142.06 −0.05 144-154 1431.72 1431.72 0.00 1431.76 1431.67 0.08 345-3652328.64 2328.64 0.00 2328.57 2328.64 −0.07  345-367* 2542.60 2542.340.26 2542.67 2542.47 0.20 366-378 1568.81 1568.70 0.11 1568.88 1568.780.10 368-373 807.97 807.95 0.02 807.94 807.88 0.06 368-376 1079.251079.30 −0.04 1079.38 1079.30 0.08 368-377 1242.49 1242.40 0.09 1242.481242.43 0.05 368-378 1355.82 1355.68 0.14 1355.70 1355.73 −0.03 368-3791469.55 1469.56 −0.01 1469.63 1469.57 0.06 368-380 1583.09 1583.10 −0.011583.04 1583.03 0.01 369-378 1208.29 1208.25 0.03 1208.33 1208.30 0.03397-408 1295.45 1295.47 −0.01 1295.41 1295.36 0.05 397-411 1643.121643.04 0.08 1643.03 1642.98 0.05 397-412 1756.26 1756.08 0.18 1756.181756.04 0.14 405-411 857.06 856.97 0.09 857.09 857.02 0.07 423-4351434.94 1434.82 0.11 1434.96 1434.78 0.18  423-436* 1598.08 1597.89 0.201598.27 1598.06 0.21  424-439* 1812.55 1812.27 0.28 1812.76 1812.37 0.39 423-439* 1869.57 1869.29 0.28 1869.79 1869.41 0.38 424-435 1377.851377.69 0.16 1378.16 1377.93 0.22  424-436* 1541.12 1540.90 0.21 1541.201541.00 0.20  425-439* 1665.29 1665.04 0.26 1665.55 1665.22 0.33 451-463* 1585.74 1585.51 0.23 1585.77 1585.53 0.24 618-641 2828.042827.98 0.06 2827.98 2827.89 0.10 621-629 989.16 989.14 0.02 989.27989.28 −0.01 621-641 2498.60 2498.59 0.01 2498.65 2498.53 0.12

The H/D exchange results summarized in Table 19 indicate that the threeregions corresponding to amino acids 345-367, 423-439, and 451-463 ofSEQ ID NO:498 are protected from full off-exchange by H4H1821N bindingto ErbB3 after on-exchange. Significantly, all three regions are locatedwithin domain III of the ErbB3 extracellular domain. Therefore, thisExample suggests that antibody H4H1821N binds a discontinuous epitopewithin domain III of the human ErbB3 extracellular domain consisting ofthese three amino acid segments or otherwise results in protection ofthese residues from H/D exchange (e.g., via conformational change orallosteric effects upon antibody binding).

Example 12 Clinical Trial of an Anti-ErbB3 Antibody in Combination withErlotinib or Cetuximab in Patients with Advanced Colorectal Cancer(CRC), Non-Small Cell Lung Cancer (NSCLC) or Head and Neck Cancer(SCCHN)

A clinical trial is conducted with the exemplary anti-ErbB3 antibodyH4H1821N in patients with advanced colorectal cancer (CRC), non-smallcell lung cancer (NSCLC) or head and neck cancer (SCCHN). The trial isdivided into two phases: a dose escalation phase and a safety expansionphase. In the dose escalation phase, all patients are initiallyadministered H4H1821N intravenously (IV) at a dose of 3, 10 or 20 mg/kg.Following the initial dose of H4H1821N, the treatment regimen ismodified based on cancer type: NSCLC patients begin a regimen of 150 mgErlotinib once daily in combination with H4H1821N at 3, 10 or 20 mg/kgIV, once every 14 days; CRC and SCCHN patients begin a regimen ofCetuximab 250 mg/m² IV once a week in combination with H4H1821N at 3, 10or 20 mg/kg IV, once every 14 days. In the safety expansion phase, NSCLCpatients receive H4H1821N (at the recommended Phase 2 dose) IV onceevery 14 days in combination with 150 mg Erlotinib once daily; CRC andSCCHN patients receive H4H1821N (at the recommended Phase 2 dose) IVonce every 14 days in combination with Cetuximab 250 mg/m² IV once aweek.

It is expected that combination therapy comprising the anti-ErbB3antibody H4H1821N and Erlotinib or Cetuximab will provide observableclinical improvements in patients with NSCLC, CRC and/or SCCHN to agreater extent than monotreatment with Erlotinib or Cetuximab alone.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1-21. (canceled)
 22. A method for inhibiting tumor growth, the methodcomprising administering to a subject afflicted with a tumor apharmaceutical composition comprising an antibody or antigen-bindingfragment thereof that specifically binds ErbB3, wherein the antibody orantigen-binding fragment interacts with amino acids 345-367 of SEQ IDNO:498, amino acids 423-439 of SEQ ID NO:498, and amino acids 451-463 ofSEQ ID NO:498, as determined by hydrogen/deuterium exchange.
 23. Themethod of claim 22, wherein the tumor is selected from the groupconsisting of a renal tumor, a pancreatic tumor, a breast tumor, aprostate tumor, a colon tumor, a gastric tumor, an ovarian tumor, a lungtumor, and a skin tumor.
 24. The method of claim 22, further comprisingadministering to the subject a second therapeutic agent.
 25. The methodof claim 24, wherein the second therapeutic agent is an anti-EGFRantibody.
 26. The method of claim 25, wherein the anti-EGFR antibody iscetuximab or panitumumab.
 27. The method of claim 24, wherein the secondtherapeutic agent is a small molecule inhibitor of EGFR.
 28. The methodof claim 27, wherein the small molecule inhibitor of EGFR is erlotinibor gefitinib.
 29. The method of claim 24, wherein the second therapeuticagent is an anti-HER2 antibody.
 30. The method of claim 29, wherein theanti-HER2 antibody is trastuzumab.
 31. The method of claim 24, whereinthe second therapeutic agent is an anti-ErbB4 antibody.
 32. The methodof claim 24, wherein the second therapeutic agent is an anti-EGFRvIIIantibody.
 33. A method for inhibiting tumor growth, the methodcomprising administering to a subject afflicted with a tumor apharmaceutical composition comprising an antibody or antigen-bindingfragment thereof that specifically binds ErbB3, wherein the antibody orantigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 and LCDR3 domains comprising the amino acid sequences,respectively, of SEQ ID NOs: 324, 326, 328, 332, 334 and
 336. 34. Themethod of claim 33, wherein the antibody or antigen-binding fragmentthereof comprises a HCVR/LCVR amino acid sequence pair comprising SEQ IDNOs: 322/330.
 35. The method of claim 33, wherein the tumor is selectedfrom the group consisting of a renal tumor, a pancreatic tumor, a breasttumor, a prostate tumor, a colon tumor, a gastric tumor, an ovariantumor, a lung tumor, and a skin tumor.
 36. The method of claim 33,further comprising administering to the subject a second therapeuticagent.
 37. The method of claim 36, wherein the second therapeutic agentis an anti-EGFR antibody.
 38. The method of claim 37, wherein theanti-EGFR antibody is cetuximab or panitumumab.
 39. The method of claim36, wherein the second therapeutic agent is a small molecule inhibitorof EGFR.
 40. The method of claim 39, wherein the small moleculeinhibitor of EGFR is erlotinib or gefitinib.
 41. The method of claim 36,wherein the second therapeutic agent is an anti-HER2 antibody.
 42. Themethod of claim 41, wherein the anti-HER2 antibody is trastuzumab. 43.The method of claim 36, wherein the second therapeutic agent is ananti-ErbB4 antibody.
 44. The method of claim 43, wherein the secondtherapeutic agent is an anti-EGFRvIII antibody.